We propose to continue our studies on the development of metallic nanostructures for rapid high-sensitivity diagnostics, and in particular arrays for cardiac markers without amplifications steps. We selected cardiac markers as the target analytes because of their strong medical importance. Coronary heart disease (CHD) is the leading cause of mortality in developed countries including the USA. Each year an estimated 8 million patients admit acutely to emergency rooms with symptoms suggestive of ischemic heart disease and myocardial infarction (MI). Rapid diagnosis is essential either to initiate rapid treatment with anti-coagulants or to determine the symptoms are non-threatening. Towards this direction our approach combines the emerging sciences of plasmonics and nano-optics to increase the sensitivity of fluorescence for clinical testing. During the present three year award cycle, we have accomplished a majority of the previously proposed Aims for testing of metallic structures including metal island films with heterogeneous distribution of particle sizes, continuous metal films, well-ordered nanoparticle arrays fabricated by e-beam lithography and nanohole arrays by a focused ion beam (FIB). Using these structures we have reached the needed clinical sensitivity goals for myoglobin, and are within a factor of 10 of the sensitivity needed for B-type natriuretic peptide (BNP) and cardiac troponin I (cTnl), without using biochemical amplification steps. We note that the cTnl requires a low detection limit of 0.22 pM. These results indicate the high potential of using metal nanostructures for clinical assays. In the current proposal we focus on the development of metallic structures which can provide an additional 100-fold increase in sensitivity. We selected this goal to insure these structures meet or exceed the clinical requirements. We will focus on nanostructures which can be easily prepared and introduced into clinical laboratories. For these reasons we propose to develop structures which can be prepared using bottom-up batch chemical methods, without the need for top-down nanofabrication technology. We propose the following: 1. Develop and optimize multi-layer substrates consisting of metal particles, a dielectric spacer and a continuous metal film for high sensitivity clinical assays. The substrates will be tested for reproducibility and stability under clinical assay conditions. 2. Use these metallic nanostructures for cardiac markers assays for myogloblin, cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP) which satisfy the clinical needs without amplification steps. The proposed layered nanostructures provide opportunities for high fluorescence enhancements and background rejection. We will compare the results with clinical samples and known standards. 3. Develop substrates for the next generation of assays using hydrogel waveguides on metallic layers. These structures offer the opportunity for efficient directional emission and wavelength separation in a simple multi-layer device.